Each time a cell divides, it must duplicate all its DNA efficiently and accurately. In order for replication to occur in a timely manner on the large eukaryote chromosomes, mechanisms which allow replication to initiate at multiple locations are required. In the yeast these sites are determined by binding of the Origin Recognition Complex, ORC to conserved origin sequences spaced along the chromosome. Functional origin sequences have yet to be identified in metazoan cells. Using extracts derived from Xenopus eggs we have developed an in vitro cell free nucleus free system for investigating DNA replication. Using this system, we find little evidence for sequence specific initiation. Rather it appears that efficient replication can initiate at most if not all sites. In this proposal, the PI intends to extend these studies by directly measuring the affinity of ORC and Cdc6 initiation factors to different DNA substrates and to determine whether ATP hydrolysis significantly modifies these affinities. These studies should resolve both whether recognition of specific sequences by ORC is an important element of metazoan replication. If ORC can bind and initiate replication at any DNA sequence, then it is essential that cells have mechanisms for ensuring that initiation sites are established at regular distances from each other. The Investigator's data suggests that such mechanisms exist. In this proposal, they describe experiments directed at verifying their existence and characterizing how they function to establish spacing between initiation sites. It has also been shown in metazoans, multiple contigous initiaton complexes are organized into discrete DNA domains and they activate replication synchronously during S-phase of the cell cycle. They have shown temporal activation of replication within a domain is dependent on cdk2 kinase activity. They intend to determine whether this is a direct effect of cdk on controlling domain activation through loading cdc45 protein onto DNA during the assembly of replication initiation sites. To successfully complete DNA replication in a reasonable period requires that many initiation sites be used. These sites or PCRs consist of ORC, cdc6, MCM, cdc7 and cdc45 proteins. With respect to how a cell might know when enough of these sites have formed the Investigator's have shown that PRC formation activates the degradation of Xic1, activating cdk2- cyclin E kinase an activator of replication. Accumulation of PRCs above a threshold number activates a pathway which causes replication to initiate. They describe experiments designed to characterize the molecular details of this new checkpoint. Succesful completion of studies described should generate a better understanding of how replication initiation sites are established, how they are spaced, how they are organized into cooperative units, and how establishment of a critical number of potential replication sites activates a signal driving the G1 to S-phase transitions.